Hematopoietic stem cells (HSCs) exist as rare populations within blood-forming tissues that both self-renew and generate all blood cell lineages for life. HSCs are born in the embryo, but relatively little is known regarding their ontogeny and early function. This is due, in large part, to these events occurring in utero in the mammalian fetus. In contrast, the zebrafish, with externally fertilized and transparent embryos, presents an ideal model system to observe directly and manipulate genetically vertebrate HSC formation and function. I have recently pioneered prospective isolation approaches, transplantation technology and in vivo imaging for the study of zebrafish HSCs. With fluorescent transgenes targeted to hematopoietic precursors, these approaches will now be used in combination for unprecedented study of the genesis of HSCs. We will perform multiplex fluorescent in situ hybridization (FISH) to dissect the molecular signatures of both HSCs and their microenvironmental niche. In addition to these static analyses, we will image the behavior of HSCs by confocal time lapse microscopy in living, multicolor transgenic animals to witness the birth of HSCs for the first time. Putative HSC subsets will be prospectively isolated by flow cytometry and tested functionally by transplantation into zebrafish blood mutants. Finally, a precise characterization of the molecular determinants and in vivo behavior of embryonic HSCs will provide the framework to systematically dissect the mechanisms of HSC support by the niche. Insight into the genetic regulation of embryonic HSC function will be translated to the adult hematopoietic system. Novel findings regarding the environmental interactions of HSCs will ultimately be examined for conservation in mammalian systems. The translation of the developmental mechanisms required to maintain and instruct stem cell function has great potential in both the in vitro generation of HSCs from human ES cells and in ex vivo manipulation of patient HSCs for cell and gene therapy.